Mechanistic Studies of the Inhibition of Insulin Fibril Formation by Rosmarinic Acid.

The self-assembly of insulin to form amyloid fibrils has been widely studied because it is a significant problem in the medical management of diabetes and is an important model system for the investigation of amyloid formation and its inhibition. A few inhibitors of insulin fibrillation have been identified with potencies that could be higher. Knowledge of how these work at the molecular level is not known but important for the development of more potent inhibitors. Here we show that rosmarinic acid completely inhibits amyloid formation by dimeric insulin at pH 2 and 60 °C. In contrast to other polyphenols, rosmarinic acid is soluble in water and does not degrade at elevated temperatures, and thus we were able to decipher the mechanism of inhibition by a combination of solution-state 1H NMR spectroscopy and molecular docking. On the basis of 1H chemical shift perturbations, intermolecular nuclear Overhauser effect enhancements between rosmarinic acid and specific residues of insulin, and slowed dynamics of rosmarinic acid in the presence of insulin, we show that rosmarinic acid binds to a pocket found on the surface of each insulin monomer. This results in the formation of a mixed tetramolecular aromatic network on the surface of insulin dimer, resulting in increased resistance of the amyloidogenic protein to thermal unfolding. This finding opens new avenues for the design of potent inhibitors of amyloid formation and provides strong experimental evidence for the role of surface aromatic clusters in increasing the thermal stability of proteins.